The Zbtb family of proteins consists of more than 40 members in mice. They are highly conserved transcriptional repressors that contain a BTB (POZ) protein-binding domain and a Kr?ppel-type Zinc Finger (ZF) DNA-binding domain. Although the function of most Zbtb members is still unknown, many Zbtb proteins have essential functions during development, and some were identified as controlling unique differentiation pathways in lymphocytes. For example, Th-POK is a master regulator of CD4 versus CD8 differentiation of T cells; Lrf (Pokemon), was shown to direct B cell versus T cell differentiation; Bcl6 is necessary for the germinal center reaction in B cells and directs the differentiation of CD4 T cells towards T follicular helper (Tfh) cells; and we and others have identified that increased expression of PLZF is necessary for the acquisition of effector functions during the differentiation of Natural Killer T (iNKT) cells, a subtype of T lymphocytes with innate and cytotoxic functions. The potential ability of different Zbtb proteins to interact with each other and to regulate transcription on multiple targets sites is expected to generate a transcriptional regulatory network of which very little is known, mostly because of the still uncharacterized immune function of most Zbtb proteins.The general goal of this project is to elucidate the contribution of specific Zbtb family members to lymphocyte differentiation and function. To this aim, two major approaches are being undertaken: a) Investigation of the function of PLZF in lymphocyte differentiation and function. iNKT cells are a subtype of lymphocytes with unique characteristics, due to their fast activation response, their cytokine-secreting capabilities and their cytotoxic functions they can shape immune responses in several pathological conditions such as in infection, autoimmunity and cancer. Previous work from us and others identified a correlation between PLZF expression and increased IL-4 production in lymphocytes, however the mechanisms by which PLZF exert these effects are currently unknown. In order to elucidate the specific mechanisms by which PLZF exert its effect on iNKT and other cells this project involves the identification of the DNA binding sites of PLZF using Chip-Seq assays as well as the study of the phenotypic and functional changes that occur in the absence or over-expression of PLZF in different immune cell types. For this purpose we use in vitro systems as well as genetically modified mice.b) Identification of novel Zbtb family members that are functionally relevant to immune regulation. A practical approach to manipulate gene expression in vitro is by the use of short hairpin RNAs (shRNA) to specifically down-regulate (knock-down) specific genes and analyze the cellular phenotype that is consequence of this down-regulation. An alternative approach to establish gene-phenotype relationships is to perform shRNA screens in vitro. These consist on the incorporation of shRNA with different specificities by different cells, isolation of the cells with a desired phenotype and identification of the shRNA clones present, which are indicative of the gene that has been knocked-down. While this approach is typically performed in vitro, the high prevalence of false positive results, due to off-target effects and artifacts of the culture, limit the success of this technique. We have then undertaken a different approach that consist on the development of conditional shRNAs that may be activated at a certain developmental stage in immune cells in vivo, therefore allowing the analysis of immune phenotypes in the mouse. This assay is performed by immune reconstitution in mice with hematopoietic progenitors that have incorporated ?inactive? shRNAs opening the possibility to perform shRNA screens in vivo. We are focusing this approach on Zbtb family members that show high expression in lymphocytes. In addition, over-expression and knock-down studies of Zbtb family members are being performed in vitro as well as the development and analysis of genetically modified mouse models for these factors, with the purpose of elucidating their contribution to a normal immune function.